The cerebral cavernous malformation (CCM) is a common hemorrhagic vascular anomaly, caused by germ line and/or endothelial somatic mutations in three known genes. It affects more than a million Americans, predisposing them to a lifetime risk of stroke and epilepsy. A lesion is alarmingly more likely to rebleed after a prior symptomatic hemorrhage, and there is currently no therapy to prevent the genesis or clinical progression of lesions. In the past 5 years, our program project has clarified key mechanisms of CCM pathogenesis through exceptional collaboration and synergy among four laboratories. These included the identification of MEKK3/KLF2/4 signaling as a primary trigger of lesion development in the setting of Ccm loss, downstream activation of RhoA kinase (ROCK), and roles of microbiome driven TLR4/CD14 signaling, intestinal mucin loss, hypoxia, dietary supplements, thrombospondin-1, thrombomodulin and clonal expansion of mutated endothelium. The Scientific Core at U Chicago optimized the high throughput assessment of lesion burden using micro-CT, and the quantification of chronic hemorrhage using densitometry of non-heme iron Perls staining. We developed familiarity with data structure for optimal sample sizes and statistics, and a discipline of prospective articulation of outcomes and blinding in the various experiments. Protocols were harmonized, and models, specimens and data were shared across projects and sites. The Core produced transcriptomic libraries of mutated cells, C. elegans, and neurovascular units from human and murine lesions with various genotypes and at different stages of development. We streamlined the collection and distribution of genotyped human CCM lesions from excised surgical specimens to the project sites. And we assessed the peripheral blood of human subjects for protein levels and microRNAs related to the signaling aberrations. Yet critical knowledge gaps remain, as pilot data has motivated hypotheses about the roles of Ccm deficient endothelium in lesion genesis versus maturation, PIK3 signaling, ADAMTS proteolysis of versican, and activated protein C anticoagulant and cytoprotective pathways. Core services shall continue, further enhanced by new techniques aimed at facilitating single-cell RNA and DNA analyses from lesions, morphometric assessment of acute hemorrhage (as a clinically relevant phenotypic feature distinct from chronic bleeding), and micro-sampling and processing for plasma biomarker discovery in mice. Each of the projects will be assisted by and take advantage of the proposed Phenotyping, Human Tissue and Biomarkers Core, aimed at (1) phenotype assessment and human lesion dissection, and (2) biomarker discovery and validation. The new hypotheses being probed will identify novel therapies, and the resulting biomarkers will facilitate risk stratification and disease monitoring in patients. In a strategic sense, the proposed Core will continue to enhance collaboration and synergy among the project sites, ensure rigor, and facilitate innovation. The Core shall allow cross-validation between mouse and human samples, further leveraging the translation of discoveries to human subjects.